Higher eukaryotes perform a variety of post-translational modifications, including methylation, sulfation, phosphorylation, lipid addition and glycosylation. Such modifications may be of critical importance to the function of a protein. Secreted proteins, membrane proteins, and proteins targeted to vesicles or certain intracellular organelles are likely to be glycosylated.
N-linked glycosylation is a form of glycosylation involving addition of oligosaccharides to an asparagine residue found in recognition sequences (e.g., Asn-X-Ser/Thr) in proteins. N-linked glycoproteins contain standard branched structures, which are composed of mannose (Man), galactose, N-acetylglucosamine (GlcNAc) and neuramic acids. Protein N-glycosylation typically originates in the endoplasmic reticulum (ER), where an N-linked oligosaccharide (e.g., Glc3 Man9 GlcNAc2) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate Asparagine (Asn) of a nascent protein. This is an event common to all eukaryotic N-linked glycoproteins. There are two major types of N-linked saccharides: high-mannose oligosaccharides, and complex oligosaccharides.
High-mannose oligosaccharides typically include two N-acetylglucosamines with many mannose residues (e.g., greater than 4). Complex oligosaccharides are so named because they can contain almost any number of the other types of saccharides, including more than the original two N-acetylglucosamines. Proteins can be glycosylated by both types of oligosaccharides on different portions of the protein. Whether an oligosaccharide is high-mannose or complex is thought to depend on its accessibility to saccharide-modifying proteins in the Golgi apparatus. If the saccharide is relatively inaccessible, it will most likely stay in its original high-mannose form. If it is accessible, then it is likely that many of the mannose residues will be cleaved off and the saccharide will be further modified by the addition of other types of group as discussed above.
After an oligosaccharide chain has been added to a protein, the three glucose and one mannose residues are removed by three different enzymes in a fixed order. This event occurs in the ER and is a signal that the protein can be transported to the Golgi for further processing. After the processing in the ER, the high-mannose type oligosaccharide is formed. The three glucose residues and one specific alpha-1,2-linked mannose residue are removed by specific glucosidases and an alpha-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man8 GlcNAc2. The protein with this core sugar structure is transported to the Golgi apparatus where the sugar moiety undergoes various modifications.
In mammalian cells, the modification of the sugar chain proceeds via 3 different pathways depending on the protein moiety to which it is added. The three different pathways are: (1) the core sugar chain does not change; (2) the core sugar chain is changed by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removing the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; or (3) the core sugar chain is first converted into Man5 GlcNAc2 by removing 3 mannose residues with mannosidase I; Man5 GlcNAc2 is further modified by adding GlcNAc and removing 2 more mannose residues, followed by sequentially adding GlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem. 54: 631-664 (1985); Chiba et al., J. Biol. Chem. 273: 26298-26304 (1998)).
The oligosaccharide content of recombinant proteins can affect the safety and efficacy of therapeutic glycoproteins. Accordingly, methods for controlling the oligosaccharide content, particularly the mannose content, of such glycoproteins would be beneficial.
The high mannose content of glycoprotein compositions, particularly therapeutic antibodies, can significantly affect the safety and efficacy of such proteins during therapeutic use. Without being bound by a particular theory, evidence suggests that high-mannose glycoproteins are cleared from circulation faster than their low mannose counterparts due to, for example, mannose receptors on macrophages and dendritic cells. Additionally, high mannose glycoproteins are expected to be more immunogenic. Accordingly, it is desirable to produce therapeutic glycoproteins such as, for example, therapeutic antibodies, having low mannose content.
The present inventors solves this need in the art by providing methods for modulating (e.g., controlling or reducing) the mannose content of recombinantly produced proteins and peptides.